Project Summary/Abstract Sjgren's syndrome (SS) is a systemic autoimmune disease that most commonly targets the exocrine glands and is characterized by persistent dry eyes and mouth as well as extra-glandular involvement. Salivary gland lymphocytic infiltrates are a pathological finding in the disease. There are B cell expansions, hyper- reactivity and antibody formation in exocrine glands of SS patients. Some patients have glandular ectopic germinal center formation, as well as the presence of Type II B cells (T2) phenotypically similar to marginal zone (MZ) B cells in the spleen serving as a checkpoint for deletion and are important in the induction/loss of tolerance. Patient serum commonly contains autoantibodies to Ro (SS-A) and La (SS-B), and the number of anti-Ro and -La specific B cells in salivary infiltrates correlate with antibody titer in the serum. Other specificities are present, including those towards muscarinic 3 receptor (M3R). The evidence suggests antibodies targeting M3R, important in para-sympathetic signaling, may induce glandular dysfunction. We, and others have demonstrated that IgG from affected individuals, when injected into nave mice, can transfer disease as manifested by salivary flow impairment. Thus, the evidence strongly supports the premise that B cells infiltrating the salivary glands of SS patients not only make autoantibodies that are in part responsible for glandular dysfunction but are also a source of some autoantibodies in the sera. This proposal will test the hypothesis that this is the case, and will address the pathogenic mechanisms underlying this dysfunction. In Aim 1, using the latest cutting-edge techniques, we will sequence the V-regions and produce monoclonal antibodies (mAb) from salivary gland plasmablasts, and compare them to the autoantibody repertoire found in the serum of the same patients, using high-resolution Orbitrap mass spectrometric Ig protein sequencing. Thus, we will determine whether anti-Ro in the circulation is produced by antibody-secreting cells in the salivary glands. Given that anti-Ro clonotypes are turned over regularly, determining that this turnover involves the B lymphocytes in the exocrine glands will be an important insight into the pathogenesis of the disease. We have demonstrated that some patients have clonally expanded B cells infiltrating the gland, while other patients do not. In Aim 2 we will determine the correlates and pathophysiology of clonally expanded B cells infiltrating the salivary glands. We hypothesize that clonally expanded B cells will be related to other immunological features in the gland and may identify populations or microenvironmental influences that drive germinal center formation, B cell proliferation and autoantibody production. We have in hand SS mAb that bind and block M3R activation, and thus, may be involved in the pathogenesis of the disease. In Aim 3 we will define the nature of pathogenic mAbs that block salivary flow, and determine if they can be inhibited. We hypothesize that pathogenic mAb will bind distinct M3R epitopes compared to non-pathogenic mAb and representation of the epitope as an inverse D-amino acid peptide will have a blocking action in our mouse model.